Cellular Microbiaxial Stretching Assay for Measurement and Characterization of the Anisotropic Mechanical Properties of Micropatterned Cells

Rothermel, Taylor M.; Cook, Bernard L.; Alford, Patrick W.

doi:10.1002/cpz1.370

Cited by 2 publications

(3 citation statements)

References 70 publications

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“…Traction force microscopy (TFM) was executed by the construction and micropatterning of polyacrylamide gel constructs as previously described 13,[89][90][91] . Briefly, stamps were made from PDMS from a master silicon wafer that was fabricated using photolithography.…”

Section: Traction Force Microscopy Of 2d Co-culture CMmentioning

confidence: 99%

“…The TFM measurements were analyzed in ImageJ as previously described 13,[89][90][91] . The cellinduced bead displacement between the cell-attached and cell-free images at each time point was calculated using a particle image velocimetry algorithm in ImageJ.…”

Section: Traction Force Microscopy Of 2d Co-culture CMmentioning

confidence: 99%

“…After a stabilization period of 2-5 minutes, EHT were excited using two continuous-excitation green lasers (532 nm, 1 W; Shanghai Dream Lasers Technology, Shanghai, China). Fluorescence intensity was captured for 10 seconds using high-resolution cameras (14-bit, 80 × 80-pixel, Little Joe, RedShirt Imaging, SciMeasure, Decatur, GA) at a rate of 500 frames per second 13,[89][90][91] .…”

Section: Optical Mapping Of Ehtsmentioning

confidence: 99%

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Progressive developmental cues from multiple cardiac cell types promote cardiomyocyte maturation

Ogle,

Givens,

Andebrhan

et al. 2024

Preprint

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Accumulating evidence in vitro indicates that the exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to adult post-natal environmental conditions is not sufficient to attain maturation. Here we instead mimic the progressive embryonic ventricular environment via co-culture of hiPSC-CM with epicardial cells and epicardial-derived cells in two-dimensional (2D) direct co-culture and 3D engineered heart tissues (EHT). When epicardial cells were combined with CM in 2D direct co-cultures the percentage of proliferating CM increased at the same time as stark electrophysiologic improvements in calcium handling. Notably, no increase in single-cell CM force generation was observed, as determined using traction force microscopy. Single-cell transcriptomics revealed a significant shift in the bulk CM population in epicardial-CM co-cultures as characterized by more fetal-like myofilament isoforms but with enhanced pathways associated with cardiac conduction and ventricular development. While co-culture of epicardial cells in the 3D-EHT model showed limited proliferation but a similar improvement in CM electrophysiologic function. These results indicate that certain aspects of CM maturation are enhanced, especially cardiac conduction, by epicardial cells while an embryonic phenotype is maintained (i.e., proliferation and myofilament isoforms). Next, consistent with developmental progression and the initiation of epicardial-to- mesenchymal transition (EMT), epicardial-derived fibroblasts were added to the EHTs containing epicardial cells. Interestingly, we observed significant myofilament maturation and increased force generation by more than an order of magnitude. Taken together, our results support the importance of mimicking developmental progression to slowly taper proliferation while gradually driving the maturation of hiPSC-CM to achieve robust in vitro function at the cell and tissue scale.

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Section: Traction Force Microscopy Of 2d Co-culture CMmentioning

confidence: 99%

Section: Traction Force Microscopy Of 2d Co-culture CMmentioning

confidence: 99%

Section: Optical Mapping Of Ehtsmentioning

confidence: 99%

See 1 more Smart Citation

Progressive developmental cues from multiple cardiac cell types promote cardiomyocyte maturation

Ogle,

Givens,

Andebrhan

et al. 2024

Preprint

View full text Add to dashboard Cite

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Continuum interpretation of mechano-adaptation in micropatterned epithelia informed by in vitro experiments

Cook

Alford

2023

Integrative Biology

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Epithelial tissues adapt their form and function following mechanical perturbations, or mechano-adapt, and these changes often result in reactive forces that oppose the direction of the applied change. Tissues subjected to ectopic tensions, for example, employ behaviors that lower tension, such as increasing proliferation or actomyosin turnover. This oppositional behavior suggests that the tissue has a mechanical homeostasis. Whether attributed to maintenance of cellular area, cell density, or cell and tissue tensions, epithelial mechanical homeostasis has been implicated in coordinating embryonic morphogenesis, wound healing, and maintenance of adult tissues. Despite advances toward understanding the feedback between mechanical state and tissue response in epithelia, more work remains to be done to examine how tissues regulate mechanical homeostasis using epithelial sheets with defined micropatterned shapes. Here, we used cellular microbiaxial stretching (CμBS) to investigate mechano-adaptation in micropatterned tissues of different shape consisting of Madin–Darby canine kidney cells. Using the CμBS platform, tissues were subjected to a 30% stretch that was held for 24 h. We found that, following stretch, tissue stresses immediately increased then slowly evolved over time, approaching their pre-stretch values by 24 h. Organization of the actin cytoskeletal was found to play a role in this process: anisotropic ally structured tissues exhibited anisotropic stress patterns, and the cytoskeletal became more aligned following stretch and reorganized over time. Interestingly, in unstretched tissues, stresses also decreased, which was found to be driven by proliferation-induced cellular confinement and change in tissue thickness. We modeled these behaviors with a continuum-based model of epithelial growth that accounted for stress-induced actin remodeling and proliferation, and found this model to strongly capture experimental behavior. Ultimately, this combined experimental-modeling approach suggests that epithelial mechano-adaptation depends on cellular architecture and proliferation, which can be modeled with a field-averaged approach applicable to more specific contexts in which change is driven by epithelial mechanical homeostasis. Insight box Epithelial tissues adapt their form and function following mechanical perturbation, and it is thought that this ‘mechano-adaptation’ plays an important role in driving processes like embryonic morphogenesis, wound healing, and adult tissue maintenance. Here, we use cellular microbiaxial stretching to probe this process in vitro in small epithelial tissues whose geometries were both controlled and varied. By using a highly precise stretching device and a continuum mechanics modeling framework, we revealed that tissue mechanical state changes following stretch and over time, and that this behavior can be explained by stress-dependent changes in actin fibers and proliferation. Integration of these approaches enabled a systematic approach to empirically and precisely measure these phenomena.

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Cellular Microbiaxial Stretching Assay for Measurement and Characterization of the Anisotropic Mechanical Properties of Micropatterned Cells

Cited by 2 publications

References 70 publications

Progressive developmental cues from multiple cardiac cell types promote cardiomyocyte maturation

Progressive developmental cues from multiple cardiac cell types promote cardiomyocyte maturation

Continuum interpretation of mechano-adaptation in micropatterned epithelia informed by in vitro experiments

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